Peptides useful in preparing hemoglobin A1c immunogens

ABSTRACT

Monoclonal antibodies specific for the glucosylated N-terminal peptide residue in Hb A 1c , a method for producing such antibodies, hybridoma cell lines secreting such antibodies and a method for their production, and immunoassay methods and reagent systems using such antibodies for the determination of Hb A 1c  in human blood samples. The monoclonal antibodies are secreted by hybridomas obtained from the fusion of a myeloma cell and a lymphocyte which has been taken from an animal, preferably a mouse, immunized with a synthetic peptide immunogen and which produces antibody specific for the glucosylated N-terminal peptide residue in Hb A 1c . The synthetic peptide immunogen comprises an N-terminal glucosylated peptide residue having at least 2 amino acid units corresponding to the N-terminus of the beta-subunit of human hemoglobin and an immunogenic carrier to which the glucosylated peptide residue is linked. The immunoassay involves treatment of the blood sample to expose the glucosylated N-terminal peptide epitope and detection thereof by binding of the specific monoclonal antibody or a fragment thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part application of applicationSerial No. 665,811, filed October 29, 1984, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the determination of the glucosylated form ofhemoglobin known as Hb A_(1c) in human blood samples. The determinationof the extent of glucosylation of hemoglobin in an individual's bloodprovides a useful index of glucose level control in diabetics. Inparticular, the present invention concerns the preparation of monoclonalantibodies which recognize specifically the glucosylated N-terminalpeptide residue in such human hemoglobin.

Patients afflicted with diabetes are incapable of metabolizing glucosein a conventional manner resulting in a build-up of glucose in theirblood and urine. Conventionally, the glucose level in such body fluidsis taken as a measure of the state of the diabetic condition which, inturn, is used as a guide for the amount of insulin or other agent to betaken or of the need to change the patient's diet.

This works moderately well except that the glucose level may fluctuatewidely in dependence upon the time and content of the last meal, thelast insulin injection, and the like. Thus, the reading will reflect aninstantaneous condition which might not truly identify the longer termstate of the diabetic condition. To circumvent the single glucosedetermination, more elaborate measurements (e.g., the 4 to 8 hourglucose tolerance tests) are used to measure the blood levels of glucosefollowing an oral administration. These latter tests are time consuming,expensive and the individual must fast during the course of the assay.

It is known that another effect of the diabetic condition is an increasein the amount of glucosylated hemoglobin (Hb) in the blood of thediabetic. Hemoglobin is a protein tetramer made up of four chains(subunits) of amino acids, each of about 143 units and having a totalmolecular weight of about 64,000. At one end of the molecule (the NH₂-terminus of the beta-subunit) there is a valine unit which can reactwith glucose. The glucosylation of hemoglobin occurs by a non-enzymaticreaction involving glucose and the alpha-amino group of valine.Following a Schiff base formation between the reactants, the glucoseundergoes an Amadori rearrangement forming 1-deoxyfructovaline. Thiscomplex is covalent and is not reversible. The glucosylation reaction isgoverned by the concentration of the reactants, e.g., hemoglobin andglucose. In a normal (non-diabetic) individual approximately 3% of thetotal hemoglobin is glucosylated. Hemoglobin tetramers with a1-deoxyfructo-valine on the N-terminus of a beta-chain are identified asbeing glucosylated or A_(1c) hemoglobin.

Glucose levels in diabetics are sufficiently high to increase the rateof glucosylation in direct dependence upon the glucose level in theblood, which reflects the severity of the diabetic condition. Withhemoglobin, the A_(1c) level is raised to about 5 to 12%. Since thecirculating life span of hemoglobin is about 120 days, a glucosylatedhemoglobin measurement will give a value which reflects an averageglucose level for that period. Notably a meal high in glucose will notbe reflected in a high glucosylated hemoglobin or serum albumin level.Thus, measurement of the glucosylated hemoglobin content gives a truerpicture of the average circulating glucose levels and thus a truerpicture of the long term condition of the patient.

One way of using this measurement has involved passing a lysed bloodsample through a boronate column, thereby selectively adsorbing theA_(1c) fraction of the hemoglobin along with some other undesiredglucosylated non-specific components. The column is washed and theA_(1c) determined spectrophotometrically. The process is complex, timeconsuming, and temperature dependent, and sometimes gives variableresults.

An alternative analytical method involved subjecting a lysed bloodsample to electrophoresis but electrophoresis is slow and expensive andrequires considerable operator skill so the test is not practical for aclinical laboratory.

Dixon in Biochem. J.(1972)129,203-208 reacted glucose withvalylhistidine, but solely for experimental purposes, no utility beingexpressed for the product.

U.S. Pat. 4,247,533 discloses an analytical technique wherein antibodiesto Hb A_(1c) were reportedly raised in a special sheep by injection ofHb A_(1c) and absorbed with nonglucosylated hemoglobin to providepolyclonal antibodies which distinguished between Hb A_(1c) andnonglucosylated Hb. Such antibodies then form the basis for a test todetermine the proportion of glucosylated hemoglobin in a sample. Thetest, however, requires an appropriately immunized sheep and antibodyabsorptions to attain the proper specificity. It is, therefore, costlyand difficult to produce specific polyclonal antibodies. The antibodypreparations produced by this absorption approach are reported to be oflow titer and affinity. The reproducibility of this approach is alsoopen to question since there are no recent reports describing its usefor the analysis of clinical samples of human hemoglobin.

Another attempt to obtain antibodies specific for Hb A_(1c) is found inU.S. Pat. No. 4,478,744. The workers in U.S. Pat. No. 4,478,744substituted a synthetic peptide immunogen for the normal hemoglobinmolecule as the immunizing agent injected into an animal which normallydoes not have Hb A_(1c) in its bloodstream, e.g., sheep. The syntheticpeptide immunogen comprised a glucosylated peptide residue having anamino acid sequence corresponding to between the first 4 to 10 aminoacids in the N-terminal hemoglobin sequence. Subsequent investigations,reported hereinbelow, have found that the sheep polyclonal antiserumraised against the synthetic peptide immunogen has no detectablespecificity for the glucosylated form, Hb A_(1c).

    ______________________________________                                        Definitions                                                                   Amino Acid         Abbreviation                                               ______________________________________                                        Arginine           Arg                                                        Aspartic Acid      Asp                                                        Glutamic Acid      Glu                                                        Lysine             Lys                                                        Serine             Ser                                                        Asparagine         Asn                                                        Glutamine          Gln                                                        Glycine            Gly                                                        Proline            Pro                                                        Threonine          Thr                                                        Alanine            Ala                                                        Histidine          His                                                        Cysteine           Cyr                                                        Methionine         Met                                                        Valine             Val                                                        Isolencine         Ile                                                        Leucine            Leu                                                        Tyrosine           Tyr                                                        Phenylalanine      Phe                                                        Tryptophan         Trp                                                        Alpha-Aminobutyric Acid                                                                          Aba                                                        ______________________________________                                    

SUMMARY OF THE INVENTION

It is accordingly an object of the present invention to provide acomparatively simple, inexpensive, reliable test for determining thelong-term blood sugar level of a patient based on the immunoassaydetermination of Hb A_(1c).

It is another object of the invention to provide such a test fordetermining the content of glucosylated hemoglobin, Hb A_(1c), in apatient's whole blood sample.

These and other objects and advantages are realized in accordance withthe present invention pursuant to which there are provided monoclonalantibodies, or fragments thereof comprising an antibody binding site,which will selectively react with the glucosylated peptide N-terminalresidue in Hb A_(1c), but not with nonglucosylated forms of such proteinso that the extent of reaction is a measure of the N-terminalglucosylated content which, in turn, is a measure or index of thediabetic condition.

It is a further object of the invention to provide cell lines which willreadily produce such monoclonal antibodies having desired specificityfor glucosylated peptide residues, specifically the residue found in HbA_(1c).

The present invention now provides the means for a highly specificimmunoassay determination of glucosylated hemoglobin in biologicalfluids such as whole blood. Monoclonal antibodies raised against thesynthetic glucosylated N-terminal peptide residues appearing in HbA_(1c) have been found to bind specifically to such residues in theglucosylated beta-subunit of hemoglobin. The antibodies can be preparedin a variety of manners following conventional antiserum and monoclonaltechniques. Principally, the antibodies are prepared against asynthetically derived immunogen comprising the desired glucosylatedN-terminal peptide residue chemically linked to an immunogenic carrier,the glucosylated peptide having at least 2, and preferably from about 5to 15, amino acid units corresponding to Hb A_(1c). The resultantmonoclonal antibodies are specific for the glucosylated syntheticpeptide and the corresponding exposed epitope for the hemoglobin A_(1c)molecule.

Monoclonal antibodies specific to Hb A_(1c) found in human blood aresecreted by hybridomas derived from fusion of myeloma cells andlymphocytes taken from an animal that had been immunized with asynthetic peptide immunogen. The synthetic peptide immunogen willpreferably be of the formula:

    [Glyco-(NH)Val-His-AA-R].sub.n Carrier

wherein Glyco-(NH)Val represents a nonenzymatically glucosylated valineresidue, His represents the second amino acid in the native beta-subunitHb sequence, AA is a bond of one or more amino acid residues, R is anappropriate linking group, Carrier is an immunogenic carrier material,and n is on the average from 1 to the number of available coupling siteson the Carrier. Linking group R can consist of any desired couplingreagent and AA can comprise one or more additional amino acid residuescorresponding to the carbohydrate-bearing N-terminus of the beta-subunitof human hemoglobin. For example, --AA-- can be selected from thefollowing amino acid sequence or any continuous fragment thereof whichbegins with the leucine unit: -Leu-Thr-Pro-Glu-Glu-Lys-. In addition,linking group R can consist of additional amino acid units not found innormal human hemoglobin but which can be conveniently added by peptidesynthesis methods and can serve as useful functional groups for couplingto the carrier material. A particularly useful linking group is-Tyr-Tyr-Cys which provides a unique thiol group for controllablycoupling the glucosylated peptide unit to carrier materials.

The present invention particularly provides a monoclonal antibody,preferably raised in mice, specific to the carbohydrate-bearingN-terminus of the beta-subunit of human hemoglobin. The antibody ischaracterized by the ability to bind a nonenzymatically glucosylatedpeptide having at least 2, and particularly from about 5 to 15, aminoacid units corresponding to the glucosylated N-terminal sequence of thebeta-subunit of hemoglobin, as well as the ability to bind to suchN-terminal sequence on hemoglobin itself.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot depicting the inhibition of Ab-3 binding to A_(1c) byglycopeptide 1 (PEPTIDE 1). Antibody was preincubated with glycopeptidebefore transfer into an A_(1c) coated mictotiter plate. The monoclonalantibody that binds to A_(1c) was detected using a secondary antibody -enzyme. The results are plotted in FIG. 1 as a percent inhibition where0% inhibition is the value obtained with no competitor. The 0--0 line isfrom an identical peptide that lacks the carbohydrate, indicating thecarbohydrate is essential for antibody binding. All points are the meanof triplicate measurements.

FIG. 2 is a plot depicting the inhibition of Ab-3 binding to A_(1c) byglycopeptide 3 (PEPTIDE 3). The competitive experiment was done asdescribed in FIG. 1.

FIG. 3 is a plot depicting the inhibition of Ab-3 and Ab-4 byglycopeptide 4 (PEPTIDE 4) for A_(1c) binding. The competitionexperiment was conducted as described for FIG. 1.

FIG. 4 is a typical standard curve using optimal assay conditions. Thewhole blood standard was prepared using different ratios of blood from adiabetic having 12.66% A_(1c) as measured by HPLC ion exchange withwhole blood from a normal donor (3.83% A_(1c)). All points of triplicatemeasurements are plotted.

FIG. 5 is a standard curve using a synthetic peptide standard. The assaywas performed as described for FIG. 4, except that instead of usingwhole blood, different amounts of synthetic glycopeptide were used asthe competitor. All values of triplicate determinations were plotted.

FIG. 6 is a plot depicting a comparison of the immunoassay method withthe boronate affinity method for donors. The mean of triplicatedeterminations are plotted for the immunoassay coordinate.

FIG. 7 is a plot depicting the results of immunizing a sheep with thesynthetic glycopeptide of Example 1(b).

FIG. 8 is a plot demonstrating that mouse monoclonal antibodies arespecific for A_(1c) hemoglobin.

DETAILED DESCRIPTION OF THE INVENTION

The monoclonal antibody of the present invention is principallycharacterized by its specificity for binding a glucosylated peptideresidue of the formula:

    Glyco--(NH)Val--His--AA--

wherein Glyco-(NH)Val and AA are as defined above. Particularlypreferred monoclonal antibodies have been found to be specific for theglucosylated dipeptide residue irrespective of the nature of AA.Antibodies with specificity requiring glucosylated peptide sequences ofgreater length are also obtainable with AA being a sequence of from 1 to12, preferably 1 to 6, amino acids corresponding to the N-terminus ofthe beta-subunit of human hemoglobin. Such specificity of the monoclonalantibody enables the specific detection of the exposed glucosylatedN-terminal peptide residue in Hb A_(1c) to the substantial exclusion ofother glucosylated peptide epitopes on hemoglobin and other proteins andpeptides native to the human bloodstream.

The glucosylated N-terminal peptide residue on the native Hb A_(1c)molecule is made accessible to the monoclonal antibody or a fragmentthereof of the present invention by appropriate denaturation ordigestion of the protein in the sample to be assayed. An underlyinghypothesis for the success of the present invention in obtainingspecific antibodies where prior art attempts have failed will now bediscussed, but its correctness should not be interpreted as beingcritical to the inventiveness of the present method.

The N-terminal sequence of the beta-subunit of human hemoglobin is quitesimilar to the corresponding sequence of mouse hemoglobin, the firstfour amino acids being identical. Secondly, mouse hemoglobin isglucosylated to approximately the same extent as human hemoglobin. Thus,in the native human hemoglobin molecule the N-terminal sequence of thebeta-subunit would not be seen by the mouse as foreign and an immuneresponse would not be expected. This is the logic of the prior artworkers who accordingly chose an animal (sheep) that has a quitedifferent hemoglobin protein sequence, and is not glucosylated in thehopes of obtaining an immune response. However, the present inventionhas revealed that when the glucosylated N-terminal residue is exposed tothe mouse immune system in the form of a synthetic peptide immunogen,the epitope is presented in a configuration to which the mouse canrespond immunologically. Through somatic cell cloning techniques,hybridomas secreting highly specific antibodies can be isolated. Thesecreted antibodies will bind to the glucosylated N-terminal peptideresidue in the native hemoglobin molecule if it has been exposedsufficiently for interaction with the combining site on the antibody.The manner of exposure of the epitope is discussed in more detail below.

Specifically, hybridoma cell lines are raised to produce antibodies onlyagainst the glucosylated portion of the hemoglobin molecule rather thanto the entire protein and such cell lines and their antibodies arescreened to identify and isolate those monoclonal antibodies which willthereafter react selectively with the glucosylated Hb A_(1c) epitope.

To produce such antibodies, a fragment of the protein chain,corresponding to the naturally occurring glucosylated peptide sequence,is coupled to a protein carrier and injected into a laboratory animal toelicit an immune response. Lymphocytes such as spleen cells from theimmunized animal are fused with myeloma cells to produce hybridomaswhich are cultured and screened for production of monoclonal antibodies.The monoclonal antibodies are screened for those selective to theglucosylated peptide epitope and the particular cell line is cloned foruse in producing further quantities of the monoclonal antibody.

To produce antibodies in the laboratory animal, e.g., BALB/c mice, ratsor the like, a glucosylated hemoglobin fragment must be either producedand isolated from naturally occurring human hemoglobin or be chemicallysynthesized and purified. The hemoglobin fragment should include the1-deoxyfructose residue and at least 2 amino acid units, preferably3,4,5 or even more, corresponding to the N-terminus of the beta-subunitof hemoglobin (valinehistidine). Advantageously it includes about 5 to15 and preferably about 7 to 10 units.

To ensure that the glucosylated peptide fragment is optimally antigenicit can be advantageously coupled to a carrier material comprising alarge immunogenic molecule such as bovine serum albumin (BSA) or keyholelimpet hemocyanin (KLH). The fragment should also carry the naturalrearranged adduct of the glucose-valine reaction which can be presentfrom the outset, as in the case of the isolated naturally occurringhemoglobin fragment, or, preferably, can be formed on the syntheticpeptide during its synthesis or before coupling the peptide to the largeprotein carrier. The carrier can be added in any manner which does notdestroy the antigenicity of the fragment.

The glucosylated fragment can be produced by chemical or enzymaticdigestion of naturally occurring Hb, e.g., A_(1c). This fragment can becoupled to a carrier using classical coupling procedures, e.g.,glutaraldehyde or carbodiimide, and the conjugate used as an immunogen.

A preferred manner of chemically synthesizing a portion of the knownhemoglobin sequence involves the addition of one or more amino acidunits (not found in the normal sequence) for optimizing its antigenicityand coupling properties. In this case, the final unit carries a thiol(SH) group by which it can be coupled to the ligand in a conventionalmanner, as by reaction with a bifunctional linking reagent such asm-maleimidobenzoyl N-sulfosuccinimide ester (MBS).

In accordance with a preferred embodiment, to the lysine end of asynthetic Hb fragment carrying the eight units NH₂-valine-histidine-leucine-threonine-proline-glutamic acid-glutamicacid-lysine-COOH there were added tyrosine, tyrosine and cysteine,resulting in an 11-unit cysteine-terminated peptide.

This can be glucosylated in conventional manner by nonenzymatic reactionwith glucose. The glucopeptide thereafter is coupled to a large carrierto produce the antigen which is administered to produce the antibodies.Lymphocytes from the animal which produce antibody to the glucosatedpeptide epitope are then fused in conventional manner to producehybridomas which are cloned and those producing monoclonal antibodies ofthe desired specification are further subcloned. The cell line(s) whosemonoclonal antibodies show the greatest selectivity for the glucosylatedepitope, as opposed to non-glucosylated Hb, are then propagated and theantibodies harvested. Reviews of such monoclonal antibody techniques arefound in Lymphocyte Hybridomas, ed. Melchers et al, Springer-Verlag (NewYork 1978), Nature 266:495 (1977), Science 208:692 (1980), and Methodsin Enzymology 73(Part B):3-46 (1981).

The antibodies can then be used in conventional manner to react withblood samples containing unknown quantities of glucosylated Hb and theextent of reaction can be compared with calibrated standards todetermine the extent of glucosylation. The read-out can be byfluorescence, by immunoassay, or the like, by joining suitably readablegroups to the monoclonal antibodies in known manner without loss oftheir binding power for the glucosylated epitope in Hb A_(1c).

Alternatively, an assay based on a reagent test strip can be run inwhich a carboxyl-carrying material such as carboxylmethyl-cellulose iscoated onto a strip of wood or plastic. Then the strip is dipped intothe lysed and denatured unknown blood sample, thereby adsorbing thehemoglobin, glucosylated or not. The strip is then dipped into asolution of the monoclonal antibodies, suitably labeled (e.g., enzyme,fluorescence, cofactors, etc.) at a site which does not interfere withbinding to the Hb A_(1c) epitope. The amount of antibody bound isdetermined by the amount of label on the strip and is an indication ofthe amount of glucosylated Hb in the unknown sample. The attachment ofthe label and its read-out are effected in conventional manner.

The immunogen used to stimulate production of appropriateimmunoglobulins in the most general sense will comprise one or more ofthe glucosylated peptide residues chemically linked to an immunogeniccarrier material. The immunogenic carrier material can be selected fromany of those conventionally known having functional groups available forcoupling to the glucosylated peptide residue. In most cases, the carrierwill be a protein or polypeptide, although other materials such ascarbohydrates, polysaccharides, lipopolysaccharides, nucleic acids, andthe like of sufficient size and immunogenicity can likewise be used. Forthe most part, immunogenic proteins and polypeptides will have molecularweights between 4,000 and 10,000,000, preferably greater than 15,000,and more usually greater than 50,000. Generally, proteins taken from oneanimal species will be immunogenic when introduced into the blood streamof another species. Particularly useful proteins are albumins,globulins, enzymes, hemocyanins, glutelins, proteins, having significantnonproteinaceous constituents, and the like. Further reference for thestate-of-the-art concerning conventional immunogenic carrier materialsand techniques for coupling haptens thereto may be had to the following:Parker, Radioimmunoassay of Biologically Active Compounds, Prentice-Hall(Englewood Cliffs, New Jersey USA, 1976); Butler, J. Immunol. Meth.7:1-24(1974); Weinryb and Shroff, Drug Metab. Rev. 10:271-283(1974);Broughton and Strong, Clin. Chem. 22:726-732(1976); and Playfair et al,Br. Med. Bull, 30:24-31(1974).

The letter "n" in the above formulas represents the number ofglucosylated residues that are conjugated to the carrier, i.e., theepitopic density of the immunogen, and will range from 1 to the numberof available coupling sites on the carrier and can be as high as 5000 inthe case of certain high molecular weight synthetic polypeptides such aspolylysine. The epitopic density on a particular carrier will dependupon the molecular weight of the carrier and the density of availablecoupling sites. Optimal epitopic densities, considering the ease andreproducibility of synthesis of the immunogen and antibody response,fall between about 10% and about 50% of the available coupling groups onthe carrier involved.

Linking group R can be essentially any convenient and stable structure.Such linking group R will usually be in the form of an aliphatic chaincomprising between 1 and about 20 atoms, excluding hydrogen, andincluding heteroatoms such as nitrogen, oxygen, and sulfur. Theglucosylated residue can be joined through a variety of groups to formlinking chain R, including methylene, ether, thioether, imino, and thelike. One skilled in the art will have a wide variety of linking groupsfrom which to choose to prepare the immunogen. Normally the glucosylatedpeptide will be prepared terminating in a functional group such asamino, carboxyl, thiol, hydroxyl, or maleimido which is active in acoupling reaction to an appropriate group in the carrier molecule.

The antibody selected for use in an immunoassay can be of anyimmunoglobulin class, e.g., IgG, IgM, and so forth, and of any subclassthereof. Normally, the antibody will be of the IgG class and if suchantibody can be used which contains an antibody combining site, e.g.,Fab, F(ab'), and F(ab')₂. The selected antibody reagent can be used inany immunoassay method for the purpose of determining Hb A_(1c) in abiological fluid. Such immunoassay methods include the more classicaltechniques such as immunodiffusion, immunoelectrophoresis, agglutinationtechniques, and complement fixation, as well as more current techniquesinvolving the use of specifically detectable labels such asradioimmunoassay and nonradioisotopic methods. The latter techniques canbe practiced in a wide variety of formats such as the competitivebinding format in which a labeled reagent is made to compete with theglucosylated analyte for binding to the antibody reagent. The amount oflabeled reagent bound to the antibody reagent, or the free-species,consisting of the labeled reagent which is not so bound, is measuredappropriately and can be functionally related to the amount ofglucosylated analyte in the sample.

In radioimmunoassays, the free-species and bound-species must bephysically distinguished or separated in order to measure the labelsince the signal generated by the label is qualitatively the same inboth species. Such a technique is known in the art as heterogeneousbecause of the phase separation requirement. Other heterogeneousimmunoassay techniques are known including enzyme-labeled immunoassays,sometimes referred to as ELISA techniques (see U.S. Pat. No. 3,654,090),and fluorescent immunoassays (see U.S. Pat. Nos. 4,201,763; 4,133,639and 3,992,631).

Fairly recently, numerous immunoassay techniques have been developedwhich obviate the separation step through the use of a label whosedetectable signal is modulated upon binding of the labeled reagent by abinding partner, e.g., antibody. Such techniques have become known ashomogeneous and are preferred for use in the present invention becauseseparations are not required and radioisotopes are not involved. Somesuch techniques are fluorescence quenching and enhancement (see U.S.Pat. No. 4,160,016), energy transfer immunoassay (see U.S. Pat. No.3,996,345), and double antibody steric hindrance immunoassay (see U.S.Pat. Nos. 3,935,074 and 3,998,943). Particularly preferred homogeneousimmunoassay techniques are those employing a label which is aparticipant in an enzyme-catalyzed reaction. Examples are thesubstrate-labeled immunoassay (see U.S. Pat. No. 4,279,992 and U.K.Patent Spec. 1,552,607), the prosthetic group (FAD)-labeled immunoassay(see U.S. Pat. No. 4,238,565), the enzyme modulator-labeled immunoassay,e.g., using inhibitor labels (see U.S. Pat Nos. 4,134,972 and4,273,866), and enzyme-labeled immunoassay (see U.S. Pat. No.3,817,837).

The monoclonal antibodies of the present invention are specific forbinding to the glucosylated N-terminal peptide residue found in HbA_(1c). The antibodies are able to bind to the epitope in the native HbA_(1c) molecule when the N-terminal epitope is appropriately exposed.Steric access to the epitope can be obtained in any effective manner.Exposure of the epitope in the intact protein is understood to beaccomplished by a physical or chemical denaturation or digestion atleast in the region of the epitope. Such denaturation or digestion canbe localized to the region of the epitope or can involve a more general,or even substantially complete denaturation of the tertiary, andadditionally the secondary, structure of the protein, or partial orcomplete digestion of the protein.

Denaturation can be accomplished in a variety of ways includingconventional treatment of the protein by physical means such as heat,sonication, high or low pH and, as is preferable, chemical denaturationby interaction with a chaotropic agent or chaotrope in solution. Usefulchaotropic agents include, without limitation, guanidine, urea, andvarious detergents such as sodium dodecylsulfate (SDS) and others,without limitation, including deoxycholate and certain bile salts,3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate, organicsolvents such as methanol, propanol, acetonitrile and certain salts suchas potassium thiocyanate. Non-ionic detergents such as Triton X-100,nonidet NP-40 and octyl-glucosides can also function as proteindenaturants. Inclusion of reagents (e.g., mercaptoethanol ordithiothreitol) that reduce disulfide bonds can be effective promotersof the denaturation process. Protein denaturation can be mosteffectively accomplished if combinations of chemical and/or chemical andphysical means are used (e.g., guanidine and heat, guanidine and SDS, orguanidine and dithiothreitol). Particularly strong chaotropes such asguanidine are most preferred.

A significant amount of Hb A_(1c) in a particular blood sample can bedenatured to expose the glucosylated epitope for antibody binding bycombining the sample, e.g., whole blood or red cell hemolysate, with anaqueous solution of the chaotrope present at sufficient concentration todenature any Hb A_(1c) in the resulting aqueous mixture. Where wholeblood is the sample, the chaotrope also serves to lyse red blood cellsto release Hb and to inactivate proteases. In the case of guanidine, theconcentration in the mixture will be preferably be greater than about1.0 molar, with about 3 molar concentration being particularly useful.The denaturation process is significantly accelerated by heating themixture for a short period of time. It has been found that attemperatures below 37° C., denaturation by the chaotrope can take fromone to several hours, whereas at temperatures above 50° C. sufficientdenaturation can be attained in a minute or less. In order to preventsignificant denaturation of the antibody and other proteinaceousreagents to be subsequently added to the mixture, the sample-chaotropemixture will normally be diluted as a separate step or by addition ofreagent solutions to a level that the chaotrope is substantiallyineffective to denature such reagents, yet will preserve the exposure ofthe epitope by preventing significant renaturation of the Hb A_(1c). Forguanidine, this preferably requires dilution to a concentration lessthan about 1.0 molar, with about 0.3 molar being particularly preferred.

Non-limiting examples of proteolytic enzymes for use in the presentinvention for digestion including trypsin, chymotrypsin,proline-specific endoprotease, pepsin and papain. In performing animmunoassay, inhibitors for the proteolytic enzymes, as are known, areadded to the assay mixture sufficient to prevent digestion ofproteinaceous assay agents.

The invention will be further described in the following illustrativeexamples wherein all parts are by weight unless otherwise expressed.

EXAMPLE 1

(a) An 11-amino acid peptide comprising the 8 N-terminal units ofbeta-hemoglobin plus two units of tyrosine plus a unit of cysteine wassynthesized according to Gutte, B. and R. B. Merrifield; J. Am. Chem.Soc., 91:2, 501(1969), giving the following peptide:

NH2-valine-histidine-leucine-threonine-proline-glutamic acid-glutamicacid-lysine-tyrosine-tyrosine-cysteine-COOH.

To glucosylate this peptide, 200 mg of this purified peptide is reactedwith 0.25 molar glucose in 20 ml of anhydrous pyridine for 48 hours atroom temperature in the dark. The mixture is dried in vacuum. Theresulting syrup is resuspended in 20 millimolar potassium phosphate, pH2.95, and purified by HPLC.

The glucopeptide-bearing fractions are dissolved in 0.1 Mtriethylammonium acetate pH 8.5 and chromatographed over Affigel-601boronate affinity resin (Biorad), whereby the glucopeptide isselectively adsorbed. The resin is washed with 0.1 M triethylammoniumacetate pH 8.5 and the glucopeptide eluted with 0.1 M triethylammoniumacetate pH 5.0. The eluate is lyophilized.

The product is resuspended in 1 ml of water, reacted with a 500 foldmolar excess dithiothreitol (to restore the SH group of the cysteine)and the reduced peptide repurified by HPLC, and lyophilized. Thisglucopeptide is stored at -20° C. under N₂ until further use.

(b) A KLH-MBS conjugate, as previously described, Lerner, R. et al,Proc. Natl. Acad. Sci. 78:3403(1981) is reacted with the product of (a)in a 2-fold molar ratio of glucopeptide to maleimide on the carrier, in50 m molar potassium phosphate, pH 7.2, for 1 hour at room temperature.

(c) The solution in (b) is mixed with equal volumes of Freund's completeadjuvant to form a water-in-oil emulsion and 200 ug of conjugate isinjected into BALB/cBy mice. The mice are boosted at 30 and 60 days,sacrificed, and their spleens used for fusion according to Kohler andMilstein, Nature 256:495(1975), producing numerous hybridomas. Thehybridomas are screened to identify those which produced monoclonalantibodies specific for the glucosylated peptide epitope.

The screening for A_(1c) specific monoclonal antibodies is conductedusing an ELISA format, where the antigen is absorbed onto polystyrenemicrotiter plates (Linbro). The antigens are purified human A_(1c) andnon-glycosylated Ao hemoglobin. The A_(1c) is purified from a red bloodcell hemolysate using two different chromatographic procedures. Thefirst purification consists of binding glycosylated hemoglobin onto aboronate affinity resin as described by Pierce Chemical Co., Rockford,Illinois, U.S.A., product no. 42,000. Typically 1 to 5 grams ofhemoglobin are applied to 100 ml boronate resin, and the bound(glycohemoglobin) fraction elutes as described by Pierce Chemical Co.,GlycoTest bulletin, product no. 42,000. The eluted glycohemoglobinfraction is equilibrated in low ionic strength buffer andchromatographed on an ion-exchange resin as described by McDonald, M. etal, J. Biol. Chem., 253: 2327-2332 (1978). The A_(1c) "peak" is analyzedby isoelectric focusing and by carbohydrate analysis using thethiobarbituric acid assay and the results confirm that this purificationproduced ultrapure A_(1c) hemoglobin in that the purified material hasboth carbohydrate and differed from normal Ao hemoglobin in isoelectricpoint. Similarly, Ao hemoglobin as purified by its property of notbinding to the boronate affinity resin and chromatographing byion-exchange as the Ao "peak" on the ion-exchange chromatographicpurification. The pure A_(1c) and Ao hemoglobins are adsorbed ontoseparate microtiter plates (2 ug per 100 microliters PBS per well)overnight at 4° C. The plates are blocked in 1% BSA in PBS for 60minutes at room temperature then washed 4 times in PBS. Supernatant fromeach hybridoma cell line is added to the A_(1c) and Ao plate andincubated at room temperature for 60 minutes. The plates are washed 4times in PBS and a secondary antibody (rabbit-anti-mouse IgG-peroxidase,Miles Laboratories, Elkhart, Indiana, U.S.A. at a 1:5000 dilution in 1%BSA in PBS) is applied and is subject to incubation for 60 minutes atroom temperature. The plate is washed 4 times in PBS and 200 microlitersof a substrate solution added (24.3 mM citric acid, 51.4 M sodiumphosphate, pH 5.3 containing 2.2 mM M o phenylenediamine and 5.2 m Mhydrogen peroxide). The reaction is terminated after 20 minutes byadding 50 microliters of 8 M H₂ SO₄ and the product of the peroxidasereaction is read at 492 mM.

From 200 starting hybridomas producing antibodies against hemoglobin,nine (9) are identified as being specific for the A_(1c) epitope,whereas 191 reacts both with A_(1c) and non-glucosylated hemoglobin.Since pre-immunized mouse serum has no detectable antibody response toAo or A_(1c) human hemoglobin by the ELISA procedure, the major immuneresponse is against the eight peptide sequence that is shared in commonwith A_(1c) and Ao. Since the synthetic peptide immunogen consists ofeight amino acid residues, of the hemoglobin sequence, the major mouseimmune response is directed against the peptide, and not thecarbohydrate (191 of the 200 hybridomas reacting both with Ao andA_(1c)) As expected, the immunized mouse serum also has broadly crossreacting antibodies reactive both with Ao and A_(1c) suggesting that nospecificity for A_(1c) is obtained unless hybridomas are screened forreactivity against A_(1c) and not against Ao hemoglobin. The preferredhybridomas producing antibodies against A_(1c) hemoglobin and notagainst Ao hemoglobin, were deposited with ATCC on Oct. 11, 1984,identified as ATCC HB 8639 and ATCC HB 8869, deposited on July 10, 1985.

(d) Identification of the peptides that compete with A_(1c) for bindingto antibody:

The following peptides are generated by enzyme digestion of theglucosylated 11-amino acid parent peptide:Glyco-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Tyr-Tyr-Cys. (GLYCOPEPTIDE 1)

All peptide fragments are purified by HPLC and quantitated by amino acidanalysis. Tryptic digestion of the parent peptide producedGlyco-Val-His-Leu-Thr-Pro-Glu-Glu-Lys. (GLYCOPEPTIDE 2)

A proline specific endoprotease produces Glyco-Val-His-Leu-Thr-Pro.(GLYCOPEPTIDE 3)

The peptide Glyco-Val-His-FAD (GLYCOPEPTIDE 4) wherein the dipeptide iscoupled to N⁶ -aminohexyl FAD and then glucosylated is made by themethod of Carrico and Johnson, U.S. Pat. No. 4,255,566 and provided byDr. Kin Yip and Dr. R. Buckler, Ames Division, Miles Laboratories,Elkhart, Indiana, U.S.A.

In a typical competition assay, each peptide 8 n moles to 8 p moles in100 μ1 PBS-7.2 mM Na₂ HPO₄, 2,8 mM NaH₂ PO₄, 127 mM NaCl, pH 7.4 isincubated with 100 μ1 monoclonal cell culture supernatant for 60 minutesat room temperature. This mixture is added to a polystyrene microtiterplate coated with 1 μg A_(1c) hemoglobin/well. If the peptide competeswith the antibody, the antibody is not free to bind to the immobilizedA_(1c). The plate is washed four times with PBS. A second antibody(rabbit anti mouse IgG coupled with horseradish peroxidase) is added for30 minutes and the plate is washed in PBS. The substrate(o-phenylenedimane 2.2 mM), and hydrogen peroxide (0.012%) are added andthe colored produced measured at 492 nm. The quantitation of the productreflects the extent of competition, e.g., no product indicates that thecompeting peptide totally blocked the antibody from binding to theimmobilized A_(1c). The results indicate that all four of the previouslydescribed glycopeptides including Glyco-Val-His-FAD are effectivecompetitors. One of the antibodies, Ab-4, is totally blocked frombinding to A_(1c) (see FIG. 1-3). Another antibody, Ab-3, is blocked byglycopeptides 1 to 3, but not by GLYCOPEPTIDE 4 (see FIG. 1-3).

Peptides lacking the carbohydrate show no competition inhibitionsuggesting that the carbohydrate is an essential component of theepitope and provides the specificity for the antibodies' recognition ofA_(lc) hemoglobin (see FIG. 1).

EXAMPLE 2 Optimal exposure of the A_(1c) epitope.

Optimal reactivity of the human A_(1c) epitope is seen followingtreatment of the native hemoglobin (in whole blood or hemolysate) withprocedures or reagents which expose the epitope to the antibodycombining site. The optimal exposure of the epitope can be accomplishedby a physical denaturation (heat, sonication, etc.), by a chemicalprocedure involving classical denaturants (urea, guanidine, SDS,protease) or by a combination of physical and chemical procedures. Mosteffective is a procedure in which whole blood (50 microliters) is addedto a 1 ml solution of 3 M guanidine hydrochloride, 10 mM Tris-HCl, pH7.4 and heated to 56° C. for greater than one minute. The resultingsample works optimally in subsequent immunoassays for the A_(1c)epitope. The solution can be diluted ten fold in buffer, effectivelydiluting the guanidine to 0.3 M, a concentration that has little if anyeffect on normal antibody-antigen interactions and enzyme activities,providing a suitable media for subsequent immunoassays.

EXAMPLE 3

(a) 1 mg of the monoclonal antibody of Example 1(c) in 0.1 molar sodiumborate buffer, pH 8.5 can be mixed with a 200-fold molar excess offluorescein isothiocyanate (FITC) and reacted for 30 minutes at roomtemperature. The fluorescein labeled monoclonal antibody can be purifiedby gel filtration.

(b) A strip (polystyrene, cellulose, etc.) carrying COOH groups isdipped into 0.5 ml of unknown denatured hemolysate, pH 7.5. The strip isrinsed with buffer at pH 7.5 and immersed into the fluorescentmonoclonal antibody of (a) in buffered solution, for 5 minutes at roomtemperature. The strip is again rinsed and the degree of fluorescence ofthe strip indicates the degree of A_(1Wlc) Hb in the unknown sample.

EXAMPLE 4 Coupling monoclonal antibody to reagent strip and its use inan immunoassay

Whatman #1 filter paper (7 cm) is placed in 20 ml ice-cold d-H₂ O andthe pH of the solution is adjusted to between 10.5-11.5 with 5 M NaOH.Solution is monitored continuously throughout the activation and the pHis maintained between 10.5-11.5 with dropwise addition of 5 M NaOH. Asmall stir bar is placed in the bottom of the beaker containing thefilter paper. The beaker is then placed in an ice filled petri dishwhich is placed on a magnetic stirrer. 1 gram of solid CnBr is added tothe beaker and this is incubated with stirring for 20 minutes (on ice).Filter paper was removed from the solution and washed in 100 ml ice-colddistilled water (d-H₂ O). It is then washed in ice-cold 0.2M Na₂ H PO₄-citric acid buffer, pH 6.8. Antibody (1 mg/ml in 0.2M Na₂ HPO₄ -citricacid buffer (PH 6.8) is added and the coupling of antibody is allowed toproceed for 1 hour. Ethanolamine (10 ml of a 1 mM solution) is added toblock unreacted sites (15 minutes) and the paper washed with phosphatebuffered saline (PBS, 10 mM NaH₂ PO₄, 140 mM NaCl, pH 7.5) to removeunreacted components.

This reagent strip is dipped into a standardized quantity of unknowndenatured hemolysate containing a labeled competitor of the antibodybinding for A_(1c) hemoglobin. A convenient competitor is theglycopeptide (GLYCOPEPTIDE no. 1) covalent coupled to horseradishperoxidase (HAPTEN-HRP). The strip is removed and rinsed with PBS. Theamount of hemoglobin bound to the strip measured (the quantity isinversely proportional to the A_(1c) in the sample and the A_(1c)hemoglobin can be quantitated by comparison to standard samples.

EXAMPLE 5 Enzyme linked immunosorbent assay for A_(1c)

A fixed volume of denatured blood hemolysate (100 μ1) is added topolystyrene microtiter plates and allowed to bind at room temperaturefor 60 minutes. The plate is washed four times in PBS containing 0.05%Tween-20 (PBST). The monoclonal antibody (coupled to horseradishperoxidase) is added (100 μ1, 1 μg/ml) in PBST and reacted for 30minutes at room temperature. The excess antibody is removed with 4washes of PBST. The substrate (o-phenylenediamine, 2.2 mM) and H₂ O₂(0.012%) in PBS are added and the reaction product measured at 492 nm.The color intensity reflects the quantity of A_(1c) present in thehemolysate when compared to standard values.

EXAMPLE 6 Radioimmoassay for A_(1c)

One hundred microliters of denatured blood hemolysate (220 n moleshemoglobin) is mixed with 7 n moles iodinatedGlyco-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Tyr-Tyr-Cys (500,000 cpm/7 nmoles). A monoclonal antibody is added in a quantity sufficient to bindto 50% of the glucosylated peptide if the blood hemolysate contains thenormal (approximately 3%) A_(1c) hemoglobin. Higher hemoglobin A_(1c)values compete for the peptide thereby reducing the total number ofcounts bound by the antibody. The antibody can be recovered byimmunoprecipitating with a second antibody (rabbit anti mouse IgG) or byadsorption onto protein A coated particles. The iodinated peptide boundto the antibody can be quantitated in a gamma isotope counter andreflects the quantity of A_(1c) present in the blood hemolysate whencompared to standards.

EXAMPLE 7 Competitive Immunoassay Using Polystyrene Beads

This competitive immunoassay is based on the use of a fixed amount ofhapten-label (as described in Example 9) that competes with A_(1c) inlysed whole blood for binding to the immobilized antibody. Since theantibody recognizes both the A_(1c) and hapten, the level of A_(1c) inthe specimen determines the amount of hapten-label that binds to theantibody. Since the antibody is immobilized, all non-bound reactants canbe removed by a simple washing step. The bound label can then bemeasured and compared to a standard for quantitation of A_(1c) in theoriginal blood samples.

The assay is developed using whole blood as the specimen and can bedivided into the steps listed below:

(1) Lysis of cells-denaturation of hemoglobin

Since the final assay requires less than 0.3 microliters of whole blood,an accurately pipettable volume of blood (5-50 μ1 from a finger stick orfrom whole blood) is diluted into a denaturing solution (3 M guanidineHCl, 10 mM Tris-HCl ph 7.5) and heated to 56° C. for 2 to 15 minutes.Lower temperatures also work, but additional time is required for thecomplete denaturation of the sample. The denaturation (a) inactivatesthe clotting mechanisms if samples are not prepared in anticoagulants;(b) lyses the red cells; (c) denatures proteases, enzymes etc., andoptimally exposes the A_(1c) epitope on hemoglobin; (d) appears toeither sterilize or inhibit the growth of microorganisms in thedenatured blood sample even if the sample is non-aseptically preparedand handled (e.g., blood from a finger stick) and (e) results in astable clinical sample that can be stored for days at room temperaturewithout effect on the final assay.

(2) Dilution and Competition An aliquot of the denatured whole blood ispipetted into a 10 fold volume of buffer containing the hapten-label.This effectively dilutes the hemoglobin to the proper concentration forthe assay and dilutes the denaturant to a low concentration so as not toperturb the antibody or enzyme activity. The antibody coated bead isthen added for a specified amount of time during which the antibodybinds either the A_(1c) hemoglobin or the hapten-HRP. (3) Wash and Read

Following the competitive incubation, the bead is washed and the labelread following the addition of an appropriate substrate. The signal isthen compared to a standard and the amount of A_(1c) present in theoriginal whole blood sample determined.

The details of the assays used are summarized below:

Bead Coating Procedure

Polystyrene beads (1/4 inch diameter with specular finish) are obtainedfrom Precision Ball Company, Chicago, Illinois, U.S.A. Lots are screenedfor beads that provided the lowest variability in multiple immunoassaydeterminations of the determinations of the same sample. Prior tocoating, beads are washed with absolute methanol followed by water. Themethanol washed seemed to significantly lower the correlation ofvariation for multiple determinations of the same sample. An antibodysolution (5 μg antibody/100 μ1 in 0.2 M sodium borate, pH 8.5, 0.02%Isodium azide) is then added to the damp beads and the beads rotatedovernight at 4° C. The beads are then washed, blocked with 1% BSA in PBScontaining 0.02% sodium azide. Typically, 500 to 1000 beads are coatedat one time and used for a period of weeks with no evidence of loss ofantibody activity. Coating experiments with radioactive antibodyindicate that 0.5 μg of antibody binds per bead.

The beads are used in this immunoassay only because of their property ofbinding relatively high amounts of protein. The hydrophobic absorptionof protein onto polystyrene is convenient, but certainly could bereplaced by one of several procedures where proteins are covalentlyattached to polystyrene, functionalized resins, or silica. Thepolystyrene can also be in the form of a tube or cuvette.

The working protocol is summarized as follows: (a) Dilute 50 microliterswhole blood into 1.0 ml denaturing solution (3 M guanidine-HCl, 10 mMTris pH 7.5), heat to 56° C., 15 minutes, dilute again 100 μ1 into 1.0ml denaturant.

(b) Add 50 microliters of the above solution to 0.5 ml phosphatebuffered saline (PBS) pH 7.5 containing hapten-HRP. The incubations,washings and enzymatic reactions are conveniently conducted in 48-wellpolystyrene tissue culture plates.

(c) Add antibody coated bead and incubate 30 minutes at ambienttemperature with rocking.

(d) Wash beads with buffer (PBS) (usually 3-1 ml changes).

(e) Add o-phenylenediamine substrate and hydrogen peroxide.

(f) Stop the reaction and read the product after 20 minutes. The aboveassay is used in establishing the clinical data described below. Thestandard curve is shown is FIG. 4. Competition using GLUCOPEPTIDE 1 isshown in FIG. 5. Evalution of normal and diabetic donors is shown inFIG. 6. The boronate affinity determination are performed exactly asdescribed by Pierce Chemcial Co. (GlycoTest, product no. 42,000).

EXAMPLE 8 Comparison of Antibody Specificity - Sheep

Polyconal vs. Mouse Monoclonal Response

A sheep is immunized, 4 sites, IM in Freund's complete adjuvant with theglycopeptide -KLH conjugate (4 mg) of example 1(b). Boost injections aredone similarly after 30 days and 60 days. The 60 day boost is inFreund's incomplete adjuvant. Preimmune serum, and immune serum istitered for its A_(1c) and Ao specificity in an ELISA assay as describedin Example 1(c). The results, (see FIG. 7) show that the syntheticglycopeptide stimulates an immune response against human hemoglobin, butthat the immunoglobulins are not specific for A_(1c) hemoglobin. Incontrast, mouse monoclonal antibodies for A_(1c) are quite specific forA when measured in the same assay (the ELISA assay of Example 1c - seeFIG. 8). Attempts to immunoaffinity purify antibody specific for Al_(c)from the sheep antiserum were not successful.

EXAMPLE 9 Preparation of Hapten-Label Conjugates

A conjuage of the GLYCOPEPTIDE 1 (HRP) was prepared The hapten-HRPconjugate was prepared by reacting 15 mg horseradish peroxidase (HRP)with a 10× molar excess of MBS (see Example 1b) in 50 mM sodiumphosphate, 1 mM EDTA, pH 7.0. The MBS-HRP conjugate was purified by gelfiltration (using the above buffer) and 0.34 mg of the glycopeptidehapten (PEPTIDE 1) was added. The final hapten-HRP conjugate waspurified by gel filtration on HPLC and was used at a dilution of1:1000-1:100,000 in the competitive immunoassay of Example 7.

It will be understood that the specification and examples areillustrative, but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:
 1. A substantially pure glycosylated peptide of theformula

    Glyco-(NH)val-His-AA-Tyr-Tyr-Cys

wherein Glyco -(NH)Val represents a nonenzymatically glycosylated valineresidue and AA is a bond of up to 13 amino acids corresponding to theglycosylated sequence in human HbA_(1c).
 2. The peptide of claim 1wherein AA is selected from the group consisting of -Leu-, -Leu-Thr-,-Leu-Thr-Pro-, -Leu-Thr-Pro-Glu-, -Leu-Thr-Pro-Glu-Glu-, and-Leu-Thr-Pro-Glu-Glu-Lys-.
 3. The peptide of claim 1 wherein AA is-Leu-Thr-Pro-Glu-Glu-Lys.